master thesis - universidade nova de lisboa arlindo tm 2015... · pom (>60 anos). endpoints do...

Master Thesis

The influence of menopausal status on bone turnover and

disease control in breast cancer patients with metastatic

bone disease treated with chemotherapy plus zoledronic

acid: an exploratory retrospective cohort study

Universidade Nova de Lisboa, Faculdade de Ciências Médicas

Northeastern University, College of Professional Studies

Arlindo Rebelo Ferreira

Medical Oncology Resident

Mentor: Luís Costa, MD, PhD

Lisbon - Boston 2015

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Abstract

Background: A recent meta-analysis showed that the adjuvant use of zoledronic acid (ZA) in

postmenopausal women with early breast cancer (BC) leads to a reduction in the risk of breast

cancer death by 17%. We investigated the effect of the hormonal status (pre [PrM] vs late post

menopause [PoM]) on bone turnover and disease control among women with BC and bone

metastases (BM) treated with ZA and chemotherapy (CT).

Methods: In this retrospective cohort study, we collected clinicopathologic variables, urinary N-

terminal telopeptide (NTX) and serum tumor marker levels from women with BC and BM treated

with CT and ZA. Patients were divided in PrM (<45 years) and PoM (>60 years). Study endpoints

were NTX, CA15.3 and CEA variation at 3, 6 and 9 months, and time to first-line CT failure and

survival. We performed multilevel mixed-effects linear regression models to assess the variation of

repeated measures and cox regression models for time to event outcomes.

Results: Forty patients were eligible for analysis (8 PrM and 32 PoM).

After introduction of ZA and CT, NTX and tumor markers declined in the overall cohort. Response

profile was similar between menopausal groups at month 3 and at later time points (p-value for

time-hormonal status interaction at month 3=0.957). Furthermore, tumor markers response profile

was also equal between groups.

Median time to first-line CT failure in PrM and PoM women was 15.2 and 17.4 months, respectively.

No significant difference between groups was found, either using a univariate analysis or after

controlling for visceral disease involvement (p=0.399 and 0.469, respectively). Likewise, no

differences in survival were found.

Conclusions: In this cohort, no differences were found in terms of NTX or tumor markers control

according to menopausal status. Similarly, no difference in time to first-line CT failure or survival

was found.

Keywords: breast cancer, bone metastases, zoledronic acid, bone remodeling markers, menopausal

status.

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Resumo

Introdução: Uma meta-análise recente demonstrou que uso adjuvante de ácido zoledrónico (AZ) em

mulheres pós-menopáusicas com cancro da mama precoce (CM) conduz a redução do risco de

morte por CM em 17%. Investigámos o efeito do estado hormonal (pré [PrM] vs pós-menopausa

tardia [PoM]) na remodelação óssea e controlo de doença em mulheres com CM e metástases

ósseas (MO) tratadas com AZ e quimioterapia (QT).

Métodos: Neste estudo de coorte retrospetivo, colhemos variáveis clinico-patológicas e

quantificámos o telopéptido N-terminal (NTX) urinário e marcadores tumorais (MT) séricos em

mulheres com CM e MO tratadas com QT e AZ. As doentes foram divididas em PrM (<45 anos) e

PoM (>60 anos). Endpoints do estudo: variação do NTX, CA15.3 e CEA nos meses 3, 6 e 9, tempo até

falência de QT de primeira-linha e sobrevivência. Quando apropriado foram usados os testes de

Wilcoxon rank-sum, modelo de efeitos lineares mistos, teste log-rank e modelo de Cox.

Resultados: Quarenta doentes foram elegíveis para análise (8 PrM e 32 PoM).

Depois da introdução de AZ e QT, os níveis de NTX e MT caíram no coorte global. O perfil de resposta

não diferiu entre grupos no mês 3 ou em tempos posteriores (valor-p para interação tempo-estado

hormonal no mês 3=0.957). Ademais, o perfil de resposta dos MT também não diferiu entre grupos.

O tempo mediano até falência de primeira-linha de QT em PrM e PoM foi de 15.2 e 17.4 meses,

respetivamente. Não foi identificada diferença significativa entre grupos, quer em análise univariada

quer após controlo para envolvimento visceral (p=0.399 e 0.469, respetivamente). Igualmente, não

houve diferenças em termos de sobrevivência.

Conclusões: Neste coorte, não foram identificadas diferenças no controlo de NTX ou MT em função

do estado menopausico. Igualmente, não foi identificada diferença no tempo até falência de

primeira-linha de CT ou sobrevivência.

Palavras-chave: cancro da mama, metástases ósseas, ácido zoledrónico, marcadores de

remodelação óssea, estado menopausico.

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List of figures and tables

Figures

Figure 1 - Interactions between bone and cancer cells in predominantly osteolytic breast cancer

lesions. Sites of action of denosumab and bisphosphonates are also noted. Bone metabolism with

resorption and formation occurs. The depicted mediators emphasize the predominant pathways.

Figure 2 – Study diagram.

Figure 3 – Mean (A), median (B) and mean log10 (C) variation of urine NTX at several time points

after diagnosis in the full cohort.

Figure 4 – Mean NTX at several time points after diagnosis according to menopausal status. Test of

differences in mean values is shown.

Figure 5 – Mean response profile of log10(NTX) at several time points after introduction of zoledronic

acid plus chemotherapy according to menopausal status. Test of interaction time - menopausal

group is shown.

Figure 6 – Mean response profile of CA15.3 and CEA at several time points after diagnosis in the full

cohort (A and B, respectively) and according to menopausal status (C and D, respectively).

Figure 7 – Mean response profile of log10(CA15.3) (A) and log10(CEA) (B) at several time points after

introduction of zoledronic acid plus chemotherapy according to menopausal status. Test of

interaction time - menopausal group is shown.

Figure 8 – Time to first-line chemotherapy failure according to menopausal status. Adjusted HR

controlling for visceral involvement is shown.

Figure 9 – Overall survival according to menopausal status. Adjusted HR controlling for HER2 status

is shown.

Tables

Table 1 – Types of bone remodeling markers.

Table 2 – Bone modifying agents.

Table 3 – Cohort baseline characteristics according to menopausal status.

Table 4 – Urine NTX at several time points from diagnosis.

Table 5 – Univariate association of baseline characteristics with time to first line chemotherapy

failure.

Table 6 – Univariate association of baseline characteristics with overall survival.

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Table of contents

Introduction ………………………………………………………………………………………………………… 6

Background and significance ……………………………………………………………………………….. 6

Study Objectives …………………………………………………………………………………………………. 14

Methods ……………………………………………………………………………………………………………… 15

Results ………………………………………………………………………………………………………………… 19

Discussion …………………………………………………………………………………………………………… 28

Conclusion ………………………………………………………………………………………………………….. 31

Acknowledgments ………………………………………………………………………………………………. 32

List of abbreviations ……………………………………………………………………………………………. 33

References ………………………………………………………….………………………………………………. 34

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Introduction This document constitutes the final thesis of the Double Master in Clinical Research from NOVA

Medical School (NOVA University) and Northeastern University.

This document is an original scientific dissertation (dissertação de natureza científica original) as per

Regulamento n.º 680/2010.

This project was presented at the “Spring Meetings 2014” (Évora, Portugal) in poster format and

was awarded a prize for the second best poster presented in the meeting.

Background and significance

a) Breast cancer epidemiology

Breast cancer (BC) is the most frequently diagnosed and the most common cause of women cancer

related death in Europe with an estimated incidence and death rate of 108.8 and 22.4 cases per 100

000 women per year, respectively.[1] More specifically, the estimated incidence and death rate in

Portugal is of 85.6 and 18.4 cases per 100 000 women per year, respectively.[1] Furthermore, breast

cancer is also the most common cause of cancer related death in women both in developed and

developing regions.[2]

In Europe and in the USA, the majority of patients currently diagnosed with BC present with early

or locally advanced disease in terms of stages of disease progression, allowing the use of therapies

with curative intent.[3] However, 5-10% of breast cancers are diagnosed with metastatic disease.

Furthermore, from those treated with curative intent, around 40% will develop disease

recurrence.[4] While locoregional recurrence might be eligible for curative interventions, those with

distant relapse are almost universally incurable.

b) Bone metastases and skeletal related events

In patients with metastatic breast cancer bone is the most common involved site, either as de novo

disease or after disease relapse. While the 5-years incidence of bone metastases in patients treated

with curative intent is around 5%, 70% of those with metastatic disease have bone involvement at

some time during disease evolution.[5] Liver, lung and less frequently the brain are also typical

target organs of breast cancer metastatic spread.

The exact mechanisms of breast cancer cells tropism to the bone is not fully understood. However,

both anatomical characteristics, as blood drainage following the Batson plexus, and molecular

features, as e.g. the chemoattractant action of bone marrow derived CXCL12 that induces tropism

of tumor cells expressing CXCR4 and CXCR7, partially explain the homing process of breast cancer

cells to the bone. After reaching the bone, Src activity seems to play a major role in supporting

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cancer cell survival in the bone marrow microenvironment. This process appears to be accomplished

by facilitating CXCL12-CXCR4-AKT signaling and by conferring tumor cells resistance to TNF-related

apoptosis-inducing ligand (TRAIL).[6] Bone metastasis are also more common in patients whose

tumors express the estrogen receptor (ER).[7] As a matter of fact, Src is active in most estrogen

receptor positive and in a minority of estrogen receptor negative tumors.[6] Furthermore, Src and

the ER seem to interact.[8] Despite this interaction, Src expression signature is more strongly

associated with bone disease than the ER per se, an association that seems independent in nature

from the ER. In line with this, even when restricting to patients with ER-negative tumors, Src

expression is still highly associated with bone metastatic development, a fact that underlines the

relevance of Src expression as, at least, a marker of activation of relevant pathways leading to a

successful adaptation of cancer cells to bone environment.[6]

Breast cancer cells further elicit a cascade of

events that lead to a profound change in bone

metabolism. Bone is under continuous

remodeling, a process accomplished by the

coupled activity of osteoblasts (bone forming

cells) and osteoclasts (bone resorbing cells).

Cancer cells reshape this equilibrium to

activate osteoblasts. This process is at least

partially accomplished through the production

of parathyroid hormone-related peptide

(PTHrp), tumor necrosis factor α (TNF-α),

interleukin 1 (IL-1), IL-6, IL-8 and IL-11 by

cancer cells.[9] Activated osteoblasts release

activator of nuclear factor κ B ligand (RANKL)

that ultimately activates osteoclasts; hence, it

induces bone resorption, which leads to the

release of growth factors entrapped in the

bone matrix (transforming growth factor-β

(TGF- β), bone morphogenetic proteins

(BMPs), insulin like growth factor (IGF) and

fibroblast growth factor).[9, 10] This last step

is the closing gap in the loop, as cancer cells will

benefit from these growth stimulation factors,

therefore leading to more bone turnover

activation: a loop referred as the vicious cycle

of bone metastases.[11]

Bone remodeling modifications ultimately

conduct to increased bone fragility[12] and

development of skeletal related events (SRE).

Figure 1 - Interactions between bone and cancer cells

in predominantly osteolytic breast cancer lesions.

Sites of action of denosumab and bisphosphonates

are also noted. Bone metabolism with resorption and

formation occurs. The depicted mediators emphasize

the predominant pathways. See text for further

details.

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Skeletal related events include a series of adverse outcomes derived from the presence of bone

metastases, namely excessive pain requiring intervention, as radiotherapy or surgery, bone fracture,

myeloid compression and hypercalcemia. The interaction between cancer and bone cells can induce

the development of several types of lesions: lytic (mostly bone degrading), blastic (mostly bone

forming) or mixed (lesions presenting a mixture of lytic and blastic features). Either of these lesions

represent a profound change in bone architecture and therefore of bone resistance to mechanical

stress. In patients without therapy aiming to control bone metabolism, a rate of approximately 4

SREs per year is expected, significantly impairing patients’ quality of life and ultimately, survival.[13,

14]

c) Markers of bone remodeling

Bone remodeling involves degradation of current bone matrix and formation of new bone matrix;

in fact, both biochemical processes lead to the release of several biochemical by-products amenable

of being quantified in, e.g., blood or urine.[15] NTX (amino-terminal crosslinked telopeptide of

collagen type I) is a by-product of collagen degradation and one of the most commonly used markers

of bone metabolism, in specific of the bone resorbing arm. On the other hand, ALP (alkaline

phosphatase) is a by-product of osteoblast activity; hence, a marker of bone formation.[15] Other

types of bone remodeling markers are listed in table 1.

Bone markers are relatively non-invasive indicators of bone metabolism and several studies tested

their role in the clinical setting. Urinary NTX has been characterized in patients with or without bone

metastases, and receiving or not bone modifying agents.[16, 17] In fact, this and other bone

remodeling markers (as CTX, carboxy-terminal crosslinked telopeptide of collagen type I) are being

explored in several perspectives in the clinical setting, namely a) to evaluate the risk of developing

bone metastases[18], b) to diagnose new bone metastases[19, 20], c) to evaluate response to

therapy/patient outcomes (SRE, bone disease progression and overall survival)[21, 22], as well as d)

as a tool to titrate the dose and frequency of administration of bone targeted agents[23]. The most

relevant use of bone remodeling markers was however during the trials that led to the approval of

new bone targeted agents as denosumab, when the variation of these markers was used as a central

study endpoint.[24] Noteworthy, its role in the clinical setting is still exploratory and, therefore, not

recommended outside clinical trials.[25]

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Table 1 – Types of bone remodeling markers.

Bone formation markers

Bone specific alkaline phosphatase (BALP)

Osteocalcin (OC)

Propeptides of type I procollagen (P1NP and P1CP)

Bone resorption markers

Amino-terminal crosslinked telopeptide of collagen type I (NTX)

Calcium

Carboxy-terminal crosslinked telopeptide of type I collagen (ICTP or CTX-MMP)

Carboxy-terminal crosslinked telopeptide of collagen type I (CTX)

Deoxypyridoline (DPD)

Hydroxyproline (Hyp), Hydroxylysine (Hyl)

Non collagenous bone matrix proteins: bone sialoprotein (BSP) and osteopontin (OP)

Osteoclast-derived enzymes: tartarate resistant acid phosphatase 5b (TRAP5b) and cathepsin k and L

Pyridinoline (PYD)

Parathyroid hormone related peptide (PTH-rp)

Vitamin D

Regulators of bone turnover

Dickkopf-1 (DKK-1)

Osteoprotegerin (OPG)

Receptor activator of NFƙB ligand (RANKL)

Sclerostin

d) Bone modifying agents

After the establishment of metastatic bone lesions two strategies are commonly implemented to

control the impact of tumor cells in the bone: the use of anti-tumor treatments and/or bone

directed treatments (osteoclast inhibitors). Anti-tumor treatments include targeted therapy (e.g.,

hormone therapy or anti-HER therapies), chemotherapy or radiation therapy. On the other hand,

bone directed therapy is composed of bone modifying agents, namely bisphosphonates (BP) and

denosumab (table 2).

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Table 2 – Bone modifying agents. NA – not applicable.

Pharmacologic Category Agents Relative potency

Bisphosphonates

Non nitrogenous-bisphosphonates

Etidronate

Clodronate

Tiludronate

1

10

10

Nitrogenous-bisphosphonates

Pamidronate

Alendronate

Ibandronate

Risedronate

Zoledronate

100

500

1000

2000

10000

Monoclonal antibodies Denosumab NA

BP are a group of pyrophosphate analogous with an intense tropism to the bone where, after

incorporation into the matrix and subsequent absorption by osteoclasts (during the process of bone

resorption), they act as potent osteoclast inhibitors (figure 1). There are two classes of BP, non-

nitrogenous and nitrogenous. While the first act by substituting the terminal pyrophosphate moiety

of adenosine triphosphate (ATP), resulting in a nonfunctional ATP molecule that competes for

functional ATP in the osteoclast energy metabolism, the latter inhibit the osteoclast enzyme farnesyl

diphosphate synthase (FDS). FDS is a critical isoprenyl diphosphate synthase or prenyl transferase

in the mevalonate pathway that is fundamental for the synthesis of sterol isoprenoids, such as

cholesterol, and non-sterol isoprenoids, such as dolichol, heme-A, isopentenyl tRNA and

ubiquinone.[26] The inhibition of isoprenoid – farnesyl diphosphate and geranylgeranyl

diphosphate synthesis by the inhibition of FDS prevents the post-translational prenylation of small

GTPase signaling proteins such as Rho, Rac, Rab, Rap and Ras leading to disruption of osteoclast

function and induction of apoptosis.[26]

On the other hand, the fully humanized monoclonal antibody denosumab acts by sequestering

RANKL (figure 1). This process limits the natural action of RANKL on the osteoclast precursor surface

receptor RANK, preventing osteoclast differentiation and ultimately bone resorption.

BP (in specific zoledronic acid, pamidronate and ibandronate), but also denosumab, are widely used

in clinical practice as tools to reduce the morbidity of bone metastases. Despite subtle differences

in relative efficacy of this agents[27, 28], major international guidelines support the use of either

zoledronic acid (ZA) or denosumab to prevent and delay SREs.[25, 29]

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e) Anti-tumoral action of bisphosphonates

BP are critical tools to prevent and delay SREs; however, pre-clinical and clinical studies are

increasingly supporting a direct and indirect anti-tumoral action of these agents. Some of the anti-

tumor mechanisms of action of BP include a direct cytotoxic action, but also other indirect

mechanisms, such as anti-angiogenesis and immune system activation.[30] While the preclinical

identification of this effects seems clear, the clinical translation of these findings is less clear, and,

so far, seems to be restricted to hormone-sensitive patients under an estrogen depleted

environment (either due to post-menopausal status or due to ovarian suppression). Furthermore,

the effect is more clear in nitrogenous-bisphosphonates, in specific for ZA.[31] Of note, more than

80% of the women with BC are post-menopausal, which opens a wide window for eventual

intervention/applicability.[3] Nevertheless, at present, available clinical practice guidelines do not

recommend the use of BP as a therapeutic tool in the adjuvant treatment of breast cancer.[25, 29,

32]

In the last decade, a group of trials documented relevant pieces of information regarding the anti-

cancer activity of BPs in the adjuvant treatment of breast cancer. The ABCSG-12 was a phase III trial

that tested the adjuvant use of ZA (4 mg every 6 month for 3 years) in 1803 pre-menopausal women

under ovarian suppression and tamoxifen or anastrazol.[33, 34] After a median follow-up of 8 years,

those patients receiving ZA had a 33% decrease in the risk of recurrence (Hazard-ratio [HR] for

disease-free survival [DFS] 0.77, 95% Confidence Interval [CI] 0.60 – 0.99; p=0.042) and a trend

towards a decrease in the risk of death (HR for overall survival [OS] 0.66, 95% CI 0.43-1.02; p=0.064).

Noteworthy, this effect was clearer in patients older than 40 years of age (HR for DFS 0.70, 95% CI

0.51 – 0.96 vs. 0.95, 95% CI 0.62 – 1.46 in women with less than 40 years of age; HR for OS 0.56,

95% CI 0.31 – 0.96 vs. 0.94, 95% CI 0.45 – 1.98 in women with less than 40 years of age).

The ZO-FAST and Z-FAST trials also tested the effect of BPs in terms of cancer related outcomes.

Despite theirs design to test as central outcomes bone health related outcomes (as variation in bone

mineral density and the occurrence of bone fracture), they also tested for cancer related outcomes

as secondary outcomes (disease recurrence). In ZO-FAST (1065 women) and Z-FAST (602 women)

postmenopausal women receiving adjuvant letrozole were randomized for upfront zoledronic acid

(4 mg every 6 months) or zoledronic acid only after bone fracture or significant decline in bone

mineral density.[35, 36] Despite de concordant results between trials in terms of improved bone

mineral density for patients receiving upfront ZA, only those patients enrolled in the ZO-FAST

derived a benefit in terms of disease recurrence (HR for DFS 0.66, 95% CI 0.44 – 0.97).[35, 36]

The AZURE trial was designed to have as a primary outcome the clinical impact of adjuvant ZA in

terms of breast cancer control related outcomes.[37] In this phase III study, 3360 pre or post-

menopausal women were randomized to standard adjuvant therapy with or without ZA (4 mg every

3-4 weeks for the first 6 months and then every 3-6 months for a total of five years). Primary

outcome was disease-free survival. At a median follow-up of around 5 years, no significant

difference in terms of DFS or OS was documented in the overall cohort. However, in a pre-planned

subgroup analysis, late post-menopausal women (those > than 5 years post-menopause) derived a

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significant benefit: 25% reduction in the risk of invasive disease or death (adjusted-HR 0.75, 95% CI

0.59-0.96) and 26% reduction in the risk of death (adjusted-HR 0.74, 95% CI 0.55-0.98).

As referred before, most of the available clinical evidence of the anti-tumor action of BPs is derived

from trials that used zoledronic acid; however, other BPs were also tested. The NSABP B-34 tested

the role of clodronate both as a strategy to test the action of this BP in terms of bone health related

outcomes (as bone mineral density), but also in terms of disease recurrence.[38] In this study, 3323

women (pre and post-menopausal) were randomized to receive standard adjuvant therapy with or

without oral clodronate (1600 mg/d for a total of 2 years). After a median follow-up of around 7

years, only those 50 years of age or older had a benefit in terms of DFS (bone and extra-bone).

However, no clear improvement in terms of OS was documented for both age groups. Pamidronate

was also tested as a strategy to reduce the risk of disease recurrence in breast cancer.[39, 40] In a

small trial Kokufu and colleagues recruited 90 patients with node positive (≥4 positive nodes) early

breast cancer treated with curative intention to receive pamidronate (n=33; 45 mg pamidronate 4

times every 2 weeks) or standard follow-up (n=57) based on personal preference.[39] After a

median follow-up of five years, a reduction in the incidence of bone metastases in the group

receiving pamidronate was found (12.1% vs. 40.4% in the control group; p=0.005). A non-significant

numerical difference favoring pamidronate was also found in terms of distant metastases (36.4 vs.

56.1%; p=0.071) and non-osseous metastases (33.3 vs. 52.6%, p=0.077). Nevertheless, no difference

regarding overall survival or disease-free survival was found. Another study testing a similar

hypothesis recruited 429 perimenopausal stage I-III breast cancer patients receiving therapy with

curative intent.[40] From these, 258 received pamidronate (15mg iv every 4 weeks and oral

pamidronate 100mg daily during adjuvant chemotherapy) while 171 didn’t. Even though patients

receiving pamidronate had a lower incidence of bone metastases (2.3% vs. 8.7% in the control

group; time of endpoint evaluation not specified) no difference was found in terms of disease-

free survival or overall survival.

Finally, a recent meta-analysis analyzed individual patient data from several randomized trials

comparing de use of BP to no BP (either placebo-controlled or open control) in terms of the risk of

recurrence and death.[31] In this study (preliminary data after analysis of 75% of 23,573 patients of

interest), a statistically significant reduction of 17% in the risk of death and distant recurrence was

found for post-menopausal women treated with BP only. Furthermore, a reduction of 35% in the

risk of bone-specific recurrence was shown, an effect also restricted to the group of post-

menopausal women. As a matter of fact, pre-menopausal women seem not to benefit from BPs in

terms of the risk of disease recurrence and death.

When taken together, an increasing body of clinical evidence is pointing towards an effect of BPs,

and more specifically ZA, as a tool to reduce the risk of recurrence and death when used in the

adjuvant treatment of post-menopausal women (5 years after menopause establishment or older

than 40 under ovarian suppression) with hormone receptor positive breast cancer. We should note

however that no current international clinical guideline is recommending the use of BPs in this

setting.[25, 29, 32]

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f) Framing the research question

Metastatic breast cancer survival has improved over time, mostly after the introduction of most

effective therapies, as taxanes, aromatase inhibitors and anti-HER agents. Other relevant gains were

achieved with more rational use of already available drugs tailored to certain risk specific groups.

BPs, and most specifically ZA, seem to have antitumor properties which depend on the systemic

hormonal environment. In fact, only late post-menopausal women (or premenopausal under

ovarian suppression therapy) seem to benefit from zoledronic acid in terms of disease recurrence

and survival. However, the available data are only derived from patients in the early breast cancer

setting, and there is no consistent evidence that BPs improve survival in metastatic breast cancer

patients nor was the effect of the hormonal environment tested in this circumstance.

In this study we aim to test if the hormonal environment significantly impacts patient outcomes, as

bone disease control, by means of the measurement of urinary NTX levels, serum levels of tumor

markers, time to first-line chemotherapy failure and overall survival in patients with metastatic

breast cancer affecting the bone and being treated with zoledronic acid and chemotherapy.

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Study Objectives To test whether the hormonal environment (pre vs. late post-menopausal) significantly impacts

bone disease control, as measured by urinary levels of NTX, serum levels of tumor markers, time to

first-line of chemotherapy failure and overall survival in patients with breast cancer affecting the

bone and being treated with zoledronic acid and chemotherapy.

a) Primary objective: To determine the impact of the hormonal environment (pre vs. post-

menopausal) on urinary levels of NTX at 3 months in patients receiving zoledronic acid and

chemotherapy;

Hypothesis: Post-menopausal patients will present a more pronounced decline in the

urinary NTX levels at 3 months when compared to pre-menopausal women.

b) Secondary objectives: To determine the impact of the hormonal environment (pre vs. post-

menopausal) in patients receiving zoledronic acid and chemotherapy on the following

parameters:

Urinary NTX levels at 6 and 9 months;

Hypothesis: Post-menopausal patients will present an increased decline in the urinary levels

of NTX at 6 and 9 months when compared to pre-menopausal women.

Serum CA15.3 and CEA levels (tumor markers);

Hypothesis: Post-menopausal patients will present an increased decline in the serum levels

of CA15.3 and CEA when compared to pre-menopausal women.

Time to first line chemotherapy failure;

Hypothesis: Post-menopausal patients will present a longer time to first line chemotherapy

failure when compared to pre-menopausal women.

Overall survival;

Hypothesis: Post-menopausal patients will present a longer overall survival when compared

to pre-menopausal women.

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Methods a) Study population

Patients with the diagnosis of metastatic breast cancer involving the bone (de novo or post-

disease recurrence), receiving zoledronic acid and chemotherapy, diagnosed and treated at

the Department of Medical Oncology from Hospital de Santa Maria – CHLN from 2000 and

2009 and with quantifications of urinary NTX available.

Evidence of breast cancer was obtained from the histopathology report, while evidence of

bone involvement was obtained from imaging.

We will exclude patients with previous diagnosis of other oncologic disease, except

contralateral breast cancer that is disease-free for more than 5 years, non-melanoma skin

cancer or pre-invasive cervix cancer. We will also exclude patients with a follow-up of less

than 3 months, with previous treatment for metastatic disease, previous treatment with

BPs and pregnant women or women with psychiatric conditions.

b) Study design and diagram

This study is a single center retrospective cohort study.

Figure 2 – Study diagram.

Women with Metastatic breast cancer

Evaluation of eligibility

Treatment with first line chemotherapy + zoledronic acid

Pre-menopausal women Post-menopausal women

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c) Data collected and study procedures

To collect the clinical variables of interest we directly extracted clinical information from medical

records, both physical and electronic.

The collected variables were the following:

Demographics: age at diagnosis, race and menopausal status;

Disease and tumor characterization: date of diagnosis, WHO performance status, AJCC

staging (T, N, number of involved lymph nodes and M), hormone receptor status, HER2

status, grade, histology, previous (neo)adjuvant chemotherapy regimen, (neo)adjuvant

trastuzumab, (neo)adjuvant chemotherapy, date of disease relapse, metastatic disease

characterization (date of metastatic diagnosis, number and localization of lesions);

Biomarkers and imaging: NTX, CEA, CA15.3, CT (thorax/abdomen/pelvis), scintigraphy,

simple radiography of the bone.

Other endpoints: date of disease progression (visceral and bone), date of introduction of

first and second line chemotherapy, date of SRE and date of death.

d) Definition of outcomes and other definitions

Biomarkers response: variation of NTX and tumor markers (CEA and CA15.3) from baseline

(before introduction of BPs) and time points of interest.

Time to first line chemotherapy regimen failure: time from introduction of BPs plus first line

chemotherapy to the introduction of second line chemotherapy regimen.

Menopausal status: Menopausal status will be assigned by age group. Women will be assigned

as pre-menopausal if less than 45 years of age; late post-menopausal women will be those with

more than 60 years of age.

e) Evaluation of urinary NTX

We used Osteomark NTx Urine ELISA (Inverness) to determine urinary NTX. The detection limit

of the NTX assay is 20 nM BCE (bone collagen equivalents units – assay value, does not include

creatinine excretion). The urinary levels of NTX in bone collagen equivalent units are expressed

as the ratio of NTX to urine creatinine excretion. Urinary NTX was defined as low (NTX<50 nmol

BCE/mmol creatinine) or high (NTX ≥ 50 nmol BCE /mmol creatinine). The cutoff values for NTX

were chosen to reflect the approximated levels of normal. However, the normal range for

urinary NTX varies according to several factors, as age, gender and endocrine environment.

While the upper level of normal (ULN) in young healthy adults is approximately 50 nmol/mmol

creatinine, after menopause, the ULN level is approximately 100 nmol BCE/mmol creatinine.[22]

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f) Statistical considerations

Descriptive statistics

Tabulation of baseline demographic, clinical, pathological and treatment characteristics

were performed according to menopausal status using Pearson’s χ2, Fisher’s exact test, t-

test or Wilcoxon rank-sum test when appropriate. Graphical representations of variation of

NTX and tumor markers through time were performed.

Analysis of outcomes

1) NTX and tumor markers (CEA and CA15.3) at baseline, 3, 6 and 9 months in the

full cohort and by menopausal status

a. Wilcoxon rank-sum test was performed to test differences between

menopausal groups at specific time points: baseline, 3, 6 and 9 months.

b. Linear mixed effects models for longitudinal data were used to model

mean variation of NTX and tumor markers over time given the

correlated nature of the outcomes (repeated measures). We used a

model with random intercepts and slopes based on the hypothesis that

individuals vary not only in their baseline level of response, but also in

terms of their changes in the mean response over time. We further

allowed the mean of intercepts and slopes to depend on the fixed effect

menopausal status category (independent variable of primary interest).

While the fixed effect parameter menopausal status informs us on how

the sample means differ between pre and post-menopausal women,

the random effect parameters represent the broad variability among

subjects. We restricted analysis to 3 time intervals, from baseline to 3,

3 to 6 and 6 to 9 months.

c. NTX and tumor markers were analyzed in absolute and log transformed

values as a strategy to focus on relative changes of biomarker at

different time points and obtain a normal distribution of biomarker

levels. Main results will be presented using the log transformed version

of the biomarkers level.

2) Time to first line chemotherapy failure and overall survival

a. The time to first line chemotherapy failure was analyzed using survival

analysis techniques. The events of interest were failure of first line

chemotherapy and death. We used the Kaplan-Meier estimator to

estimate the survival function and Kaplan-Meier plot according to

menopausal status.

b. Log-rank test was used to test the univariate difference between

survival curves of the group of pre and post-menopausal women.

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c. Cox proportional hazards model was used to test the multivariate

difference between survival curves according to menopausal status.

Outline of the model:

Outcome/dependent variable: failure of first line chemotherapy

and death;

Independent variable: menopausal status;

Covariates: estrogen receptor status, HER2 receptor status,

histologic grade, histology and concomitant visceral involvement.

Due to limitations of sample size/number of events only bivariate

models were built.

Sample size calculation

This study is of exploratory in nature, so no prior knowledge is available in terms of primary

outcome to perform a sample size calculation. Furthermore, we are constrained by a limited

set of NTX evaluations. No further NTX evaluations were performed due to funding

constrains. We will use the present results as the foundation for future assessment of

feasibility of a larger study testing the same hypothesis.

Sources of confounding and strategies to avoid confounding

The following variables were selected as potential sources of confounding: estrogen

receptor status, HER2 receptor status, histologic grade, histology and concomitant visceral

involvement. Multivariate analysis used as a strategy to overcome confounding.

g) Ethical considerations

This study was approved by the ethics committee of the Lisbon Academic Medical Center. A

waiver of the use of informed consent was granted based on the fact that urine and blood

samples had already been collected for the purpose of the evaluation of NTX and tumor markers

in the context of related studies and clinical practice, respectively. Moreover, the investigation

per se included no more than minimal risk in the form of loss of confidentiality. To overcome

this liability, several measures were taken. Some of these measures included the de-

identification of patients and subsequent re-identification using a study specific numerical study

code (p.e. 001); variables were also coded numerically (e.g., yes/no variables will be coded as

1/0, respectively) and a separated code of variables created. Moreover, electronic files were

always kept inside CHLN network or in a very limited set of other encrypted computers that had

continuous anti-virus protection. Furthermore, electronic files were always stored using an

alphanumeric password protection (either the specific file or after placing the file inside a

password protected .rar library).

of 38

Results a) Cohort characteristics

A total of 40 women were included in this study, 8 (20.0%) of which pre-menopausal and 32 (80.0%)

post-menopausal. Relevant patient’s clinical and pathologic characteristics are summarized in table

3. As a matter of fact, the groups were reasonably balanced regarding most of the selected variables.

Noteworthy, median ages are compatible with corresponding menopausal status. Furthermore,

most tumors were estrogen receptor positive, as expected from a cohort of patients with bone

disease as first site of relapse, and had a fairly good performance status, which enabled the use of

chemotherapy as a treatment option.

Table 3 – Cohort baseline characteristics according to menopausal status.

Pre-menopausal women

Late post-menopausal women

P-value

Number, % 8 (20.0) 32 (80.0) -

Age at diagnosis, median (IQR) 39.8 (35.9 – 44.2) 65.6 (60.1 – 67.8) -

Age at diagnosis of metastatic disease, median (IQR)

44.8 (40.6 – 46.5)

67.2 (62.9 – 72.9)

-

Disease-free interval (excludes stage IV patients), median (IQR)

39.7 (22.9 – 85.8)

42.6 (28.0 – 83.4)

0.366

Histology, n (%) Invasive Ductal carcinoma

Other Unknown

7 (100)

0 (0) 1 (12.5)

19 (79.2) 5 (20.8) 8 (25.0)

0.737

Estrogen receptor status Positive

Negative Unknown

7 (100)

0 (0) 1 (12.5)

21 (80.8) 5 (19.2) 6 (18.8)

0.559

HER2 status Positive

Negative Unknown

1 (20.0) 4 (80.0) 3 (37.5)

5 (27.8)

13 (72.2) 14 (43.8)

0.608

Stage IV (at diagnosis), n (%) Unknown

1 (12.5) 2 (25.0)

9 (47.4) 13 (40.6)

0.345

Visceral involvement at diagnosis of metastatic disease, n (%)

Unknown

3 (37.5)

0 (0)

4 (15.4) 6 (18.8)

0.315

Performance status, median (IQR) 0 (0 - 0) 0 (0 - 1) 0.132

Total follow-up, median (IQR) (months)

36.8 (30.0 – 53.0) 53.3 (24.9 – 62.7) 0.080

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a) Urinary NTX

The majority of patients had elevated levels of urinary NTX at baseline, which is compatible with

active disease in the bone. Moreover, when evaluating the full cohort, after the introduction of

zoledronic acid and chemotherapy (at diagnosis - baseline) a decrease of NTX is notable (table 4 and

figure 2), with 41.2% of the patients with elevated NTX at baseline reaching an NTX < 50 nmol/mmol

creatinine at month 3. Furthermore, after log-transforming NTX values, a trend for an overall decline

in mean log10(NTX) in the interval 0 – 3 months (p=0.125) and a statistically significant decline of

log10(NTX) in the interval 3 – 6 months (0.013) was found. Importantly, the proportion of patients

that were lost to follow-up in terms of urinary NTX measurements was high: while at month 6 75%

of the patients had a valid measurement of urinary NTX, at month 9 only 37.5% had. Of note, no

clear differences in terms of age, disease progression or vital status were found between lost to

follow-up and retained patients; therefore, we considered this missing information as missing

completely at random.

Table 4 – Urine NTX at several time points from diagnosis. NTX measured in nmol/mmol creatinine.

Baseline 3 months 6 months 9 months Number, % 40 (100) 39 (97.5) 30 (75.0) 15 (37.5)

Urine NTX, median (IQR) All

Pre-menopausal Post-menopausal

119.8 (66.5 – 211.1) 70.9 (43.5 – 137.2)

121.3 (71.9 – 245.0)

58.2 (25.6 – 135.9) 49.7 (32.9 – 80.0)

61.0 (25.6 – 161.9)

46.23 (20.7 – 123.9)

26.2 (12.3 – 47.2) 55.8 (24.8 – 124.8)

62.1 (32.8 – 162.9)

100.3 (50.8 – 200.7) 57.4 (31.5 – 139.3)

Elevated urine NTX, n (%) All

Pre-menopausal Post-menopausal

34 (85.0) 5 (62.5)

29 (90.6)

23 (57.5) 4 (50.0)

19 (59.4)

24 (60) 3 (37.5)

21 (65.6)

35 (87.5) 8 (100)

27 (84.4)

NTX normalization (if elevated at baseline), n (%)

All Pre-menopausal

Post-menopausal

- - -

14 (41.2) 4 (80.0)

10 (34.5)

14 (41.2) 5 (100) 9 (31.0)

4 (11.8) 0 (0)

4 (13.8)

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Figure 3 – Mean (A), median (B) and mean log10 (C) variation of urine NTX at several time points

after diagnosis in the full cohort.

When stratified by menopausal status (figure 3), post-menopausal women present persistently

absolute higher levels of mean NTX, namely at baseline and at 3 months; however, such differences

do not reach statistical significance. In fact, such differences are non-significant through all the

follow-up period. After log-transforming NTX values (figure 4), the early response (baseline to month

3) to zoledronic acid and chemotherapy is equal between groups (p=0.957). Both groups seem to

“escape” zoledronic acid plus chemotherapy control after month 6, as demonstrated by an increase

of NTX in both groups. Of note, in absolute and relative terms, urinary NTX levels seems to increase

more pronouncedly in the group of premenopausal women in this interval (figure 3 and 4); however,

this difference is non-significant (p=0.262). Moreover, when all the follow-up period is taken

together, the response profile of urinary NTX levels to zoledronic acid and chemotherapy does not

differ between pre and post-menopausal women (p=0.314).

A B

C

-100

01

00

20

03

00

40

0M

ean

NT

X

0 3 6 9Months

NTX Standard deviation

05

01

00

15

02

00

Me

dia

n N

TX

0 3 6 9Months

NTX Interquartile range

11

.52

2.5

Me

an

log

(NT

X)

0 3 6 9Months

log(NTX) Standard deviation

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11

.52

2.5

Me

an

log

(NT

X)

0 3 6 9Months

Premenopausal Postmenopausal

Standard deviation

Figure 4 – Mean NTX at several time points after diagnosis according to menopausal status. Test of

differences in mean values is shown.

Figure 5 – Mean response profile of log10(NTX) at several time points after introduction of zoledronic

acid plus chemotherapy according to menopausal status. Test of interaction time - menopausal

group is shown.

-10

00

100

200

30

04

00

Me

an

NT

X

0 3 6 9Months


Standard deviation

p = 0.957 p = 0.316 p = 0.262

p = 0.314

p = 0.187 p = 0.444 p = 0.120 p = 0.471

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b) Tumor markers (CA15.3 and CEA)

The majority of patients had elevated levels of serum CA15.3 and CEA at baseline, which is

compatible with active breast cancer. After the introduction of zoledronic acid and chemotherapy

(at diagnosis - baseline) a slight numerical decrease of both markers is identifiable (figure 5 and

figure 6); however, only a trend for decreased values through the interval 0 – 9 months is found (p

= 0.058 and 0.057, for CA15.3 and CEA respectively).

When stratified by menopausal status (figure 6), no clear differences are found between pre and

post-menopausal women in the log transformed values, either at baseline or at the following time

points, both for CA15.3 and CEA.

Figure 6 – Mean response profile of CA15.3 and CEA at several time points after diagnosis in the full

cohort (A and B, respectively) and according to menopausal status (C and D, respectively).

-200

02

00

40

06

00

80

0M

ean

CA

15

.3

0 3 6 9Months


Standard deviation

A B

C D

-200

02

00

40

06

00

Me

an

CA

15

.3

0 3 6 9Months

CA15.3 Standard deviation

-50

05

01

00

Me

an

CE

A

0 3 6 9Months

CEA Standard deviation

-50

05

01

00

Me

an

CE

A

0 3 6 9Months


Standard deviation

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Figure 7 – Mean response profile of log10(CA15.3) (A) and log10(CEA) (B) at several time points after

introduction of zoledronic acid plus chemotherapy according to menopausal status. Test of

interaction time - menopausal group is shown.

0.5

11

.5M

ean log

(CE

A)

0 3 6 9Months


Standard deviation

11

.52

2.5

3M

ean log

(CA

15.3

)

0 3 6 9Months


Standard deviation

p = 0.997 p = 0.402 p = 0.531

p = 0.762

p = 0.683 p = 0.670 p = 0.315

p = 0.478

A

B

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0

10

20

30

40

50

60

70

80

90

100

Sw

itch

ing fir

st lin

e c

he

mo

the

rap

y(%

)

17 17 11 9 7 5Post-menopause8 6 6 5 4 1Pre-menopause

No. at risk

0 5 10 15 20 25Follow-up time (months)

Pre-menopause Post-menopause

c) Time to first-line chemotherapy failure

When evaluating time to first-line chemotherapy failure, the full cohort presented a median follow-

up time of approximately 17 months (IQR 6.3 – 23.2). A total of 30 events (90.9%) were documented,

of which 8 (100%) in the premenopausal group and 22 (88.0%) in the post-menopausal group. The

median time to chemotherapy failure was of 15.3 months for pre-menopausal women and 17.4 for

post-menopausal women. In figure 7 a representation of the survival function according to

menopausal status is depicted. When evaluating the univariate association of menopausal status

and other clinical and pathological variables with change of first line chemotherapy, only the

presence of visceral involvement at diagnosis of metastatic disease was significantly associated with

time to chemotherapy switch (table 5). Furthermore, when testing the role of menopausal status

on time to first line chemotherapy failure controlling for visceral involvement a persistent non-

significant association was noted (figure 7). Despite the intention to control for other variables, due

to the lack of available observations and the fact that only concomitant visceral involvement was

significantly associated with first-line chemotherapy failure, only this covariate was included in the

model.

Figure 8 – Time to first-line chemotherapy failure according to menopausal status. Adjusted HR

controlling for visceral involvement is shown. HR – Hazard ratio; CI – Confidence interval; mo –

months; n – number.

Pre-menopause Post-menop.

Median time, mo. 15.25 17.40

Events, n (%) 8 (100) 22 (88.0)

Log-rank p = 0.399

Adjusted HR 0.72, 95% CI 0.29 – 2.77; p = 0.469

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Table 5 – Univariate association of baseline characteristics with time to first line chemotherapy

failure.

P-value

Menopausal status 0.399

Age at diagnosis of metastatic disease 0.673

Time from diagnosis to metastatic disease 0.272

Histology 0.882

Estrogen receptor status 0.867

HER2 status 0.915

Stage IV at diagnosis 0.418

Visceral involvement at diagnosis of metastatic disease

0.015

Performance status 0.240

d) Overall survival

When evaluating overall survival, the full cohort presented a median follow-up time of

approximately 49.6 months (IQR 28.4 – 60.4). A total of 34 events (85.0%) were documented, of

which 8 (100%) in the premenopausal group and 26 (81.3%) in the post-menopausal group. The

median time to death was of 30.9 months for pre-menopausal women and 52.8 for post-

menopausal women. In figure 8 a representation of the survival function according to menopausal

status is depicted. When evaluating the univariate association of menopausal status and other

clinical and pathological variables with change of first line chemotherapy, no significant associations

were found (table 6). However, HER status reached significance at 90% level of confidence, so we

decided to adjust menopausal status to this variable as well in a bivariate model. In this bivariate

model, no significant association was found as well (figure 8). As for the evaluation of time to first-

line chemotherapy failure, despite the intention to control for other variables, due to the lack of

available observations no other key variables were tested in the model.

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0

10

20

30

40

50

60

70

80

90

100

Pa

tients

aliv

e(%

)

20 18 14 6Post-menopause8 7 4 1Pre-menopause

No. at risk

0 20 40 60Follow-up time (months)

Pre-menopause Post-menopause

Figure 9 – Overall survival according to menopausal status. Adjusted HR controlling for HER2 status

is shown. HR – Hazard ratio; CI – Confidence interval; mo – months; n – number.

Table 6 – Univariate association of baseline characteristics with overall survival.

P-value

Menopausal status 0.221

Age at diagnosis of metastatic disease 0.999

Time from diagnosis to metastatic disease 0.294

Histology 0.733

Estrogen receptor status 0.618

HER2 status 0.094

Stage IV at diagnosis 0.662

Visceral involvement at diagnosis of metastatic disease

0.186

Performance status 0.377

Pre-menopause Post-menop.

Median time, mo. 30.89 52.82

Events, n (%) 8 (100) 26 (81.25)

Log-rank p = 0.221

Adjusted HR 0.63

95% CI 0.26 – 1.54; p = 0.317

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Discussion

Pre-clinical[30] and more recently clinical[31] evidence is accumulating in favor of an anti-cancer

activity of BPs, more specifically in the adjuvant treatment of post-menopausal women with breast

cancer. In the present study we intended to explore the differential anti-cancer effect of BPs

according to the hormonal environment in metastatic patients receiving ZA and chemotherapy. In

this subgroup of patients, no evidence of a differential anti-cancer activity of ZA between pre and

post-menopausal women was found when evaluating anti-cancer activity in terms of variation of

NTX (as surrogate for bone disease activity), variation of CA15.3 and CEA (as surrogate for overall

disease activity), time to first line chemotherapy failure (as surrogate for overall disease

progression) and overall survival.

BPs were developed and introduced in clinical practice as a tool to decrease the morbidity of bone

metastases given their properties as bone remodeling modulators, in specific as osteoclast

inhibitors. In this context, the primary endpoints of pivot trials were essentially centered on the rate

of SREs, time to first and subsequent SRE, improvement in pain control and general quality of life.

As a matter of fact, compared to placebo BPs reduced the SRE risk by 15% (RR 0.85, 95% CI 0.77-

0.94), with a median 28% reduction (range, 14–48%) of SREs.[41] However, no impact on overall

survival was found.[41]

Other studies tested the efficacy of BPs (zoledronic acid, pamidronate and clodronate) in the

metastatic setting with a focus on their anti-cancer activity with generally inconclusive findings.

However, based on the data derived from adjuvant studies, ours was the first study to test the

impact of menopausal status in the anticancer activity of BPs.

Two studies tested the anti-cancer activity of ZA in the metastatic setting. The MACS1295 study

(NCT01129336), previously referred as Z-ACT, tested the anti-tumoral action of ZA in patients with

HER2-negative metastatic breast cancer receiving standard first or second line therapy irrespective

of the menopausal status. In this study, patients were allocated to two different arms according to

the presence of bone metastases. Patients with bone metastases received standard therapy and ZA

(4 mg IV monthly during months 1-18), while patients without bone metastases were randomized

to receive standard therapy with or without ZA (same schedule). Primary outcome was the number

of participants with progression free survival at month 18, and secondary outcomes included the

percentage of patients with circulating tumor cells count above 5 cells per 7.5 mL of peripheral

blood, at several time points. Unfortunately, from the 150 planned patients without bone

metastases to be recruited, only 15 were included, making the interpretation of results not viable

(results available only at clinicaltrials.gov). Despite not stratifying according to the menopausal

status, this study, if properly completed, could have contributed to the improved understanding of

the clinical anti-tumor action of the zoledronic acid in the metastatic setting. Furthermore, the

BISMARK study (NCT01129336) indirectly tested the anti-tumor action of zoledronic acid.[42] In this

phase III non-inferiority study, 289 patients with bone metastases from breast cancer were

of 38

randomized to receive zoledronic acid, either as a standard fixed schedule (4mg IV over 15 minutes

once every 3-4 weeks for 24 months) or under a set of rules dependent on NTX variation. Despite

the accrual limitations that led to premature study closure, the NTX-directed schedule group had a

higher proportion of patients presenting with an SRE, thus failing to demonstrate non-inferiority of

this approach. Of note, overall survival was selected as a secondary outcome in this study. Given

that patients in the standard schedule arm had more than the double of ZA administrations, this

study can help us gain some insight on the anti-tumor action of zoledronic acid in terms of overall

survival. No stratification based on menopausal status is planned so far.

Clodronate and pamidronate (in its oral formulation) were also tested as anti-neoplastic agents, in

specific, about their ability to prevent the development of bone metastases in patients with breast

cancer without clinical evidence of bone involvement and irrespective of menopausal status.[43–

45] Despite the fact that all studies were considerably underpowered to show a difference between

groups, when compared to placebo, no difference was found in the incidence of new bone

metastases. A recent meta-analysis summarized these findings, and found no overall effect of these

agents on the incidence of bone metastases in patients with metastatic breast cancer without

clinical evidence of bone metastases (HR 0.99, 95% CI 0.67 – 1.47).[41]

So far, while the studies performed in the early setting seem to show a protective action of BPs in

terms of disease recurrence and survival in patients that are estrogen deprived (post-menopausal

or under ovarian suppression), no such evidence is available in the metastatic setting. As a matter

of fact, the role of menopausal status is still unexplored in the metastatic setting; however,

fundamental differences between the early and advanced disease settings might limit the anti-

tumoral action of BPs in latter stages. First, the burden of disease of metastatic disease is larger,

than e.g., residual disease after treatment with curative intent. The intensity of BPs anti-tumor

effect might only be clinically relevant for smaller volumes of disease, hence limiting the action in

advanced cancer. Second, the central action of BPs might be mostly relevant before metastatic

disease fully develops. In this regard, BPs seem to interfere with the vascularization of the

premetastatic niche at several tentative distant sites of metastatic development.[30] Bone medulla

is also frequently considered a sanctuary for disseminated tumor cells.[46] Similarly to the action at

premetastatic niches, BPs might change bone medulla in such a way that hampers DTC survival,

either through the anti-angiogenic action or through the action on bone cells.[30] Third, post-

menopausal women present a more active bone metabolism, which some hypothesized that might

contribute to the establishment of bone metastasis.[18] BPs block bone resorption and unbalance

bone metabolism towards bone creation. This phenomena might impair the fixation of tumor cells,

a mechanism not transposable to the metastatic setting. On the other hand, certain mechanisms

may play similar roles both in the early and metastatic settings. BPs disrupt the vicious cycle

between cancer cells and bone remodeling activation, which decreases the availability of growth

factors entrapped in the bone matrix. Likewise, the immune activation, namely through γδ T cells,

would reasonably act on both settings of disease evolution.

of 38

The present study presents important limitations which is compatible with its exploratory nature

and budget constraints. The first main limitation refers to the small sample size. Despite the

preplanned design as an exploratory study, the group of premenopausal women was considerably

small, which might not be representative of a conventional cohort of pre-menopausal patients.

Second, a relevant lost to follow-up was found at 9 months. Regardless of the intention to perform

the analysis of primary outcomes at 3 months, secondary outcomes interpretation is limited due to

this fact. Indeed, the late acceleration of urinary NTX in the pre-menopausal patients cannot be

properly valued in this context. Third, time to first line chemotherapy failure after the diagnosis of

metastatic disease was considerably long. In fact, several of these patients stopped first line

chemotherapy after a good clinical response and had a switch to hormone therapy before switching

to second line chemotherapy, as common practice in patients with hormone receptor positive

disease. The present analysis does not account for the type of hormone therapy received in the

transition to second line chemotherapy and this fact might have been a source of heterogeneity. As

a matter of fact, hormone therapy is known to interfere in bone remodeling[47, 48], and different

options of hormone therapy might change bone remodeling differently. Finally, the same threshold

of NTX normality was used both for pre and post-menopausal women for comparability reasons. It

is long known that several factors interfere with bone remodeling markers levels, such as age,

hormonal environment and gender. The persistently higher levels of NTX in post-menopausal

women at baseline and after the introduction of zoledronic acid and chemotherapy (despite all of

which non-significant) might be exclusively associated with this fact.

Noteworthy, to our best knowledge, none of the published studies so far tested the menopausal

status as a modulator of BPs action as an anti-tumor agent. In light of our findings and study

limitations this question remains unanswered.

of 38

Conclusions

In a cohort of women with breast cancer and bone metastases receiving first line chemotherapy and

zoledronic acid, no difference seems to exist in terms of urinary NTX variation at 3 months according

to menopausal status. Furthermore, no differences was found in terms of CA15.3 and CEA variation

according to menopausal status. Finally, time to first line chemotherapy failure or overall survival

seems to be similar when evaluated according to menopausal status.

Taken together, these results do not support a differential action of zoledronic acid in the control of

bone metabolism (as a surrogate of cancer cells activity) or other cancer control related outcomes

according to menopausal status. However, these findings need to be considered side with the

discussed study limitations. As a matter of fact, this body of data can only be used as preliminary

findings for future studies intending to test the anti-cancer activity of BPs according to the hormonal

environment.

of 38

Acknowledgments

I would like to acknowledge Doctor Irina Duarte for her decisively contribution to this work with the

quantification of NTX in the lab. I would also like to acknowledge Dr. Inês Vendrell for helping in the

collection of clinical information and all the personal support to complete the current Master.

Likewise, I would like to thank Irene Ferreira for the construction of figure 1 and all the enthusiasm

she always gave me on pursuing this Master. Furthermore, I would like to recognize the profound

contribution of Dr. Inês Vaz-Luís for all the mentorship around the development of this project since

its inception, as well as for all the contributions to my personal and professional development.

Moreover, I would like to thank Prof. Doctor Luís Costa for the personal inspiration and the role

model he became during my medical school years to aspire on being a physician scientist and more

recently on raising my awareness for the topic of bone metastases. He was also a cornerstone on

recruiting most of the patients analyzed. He further contributed with insightful comments to this

work and thesis. Finally, I would like to thank my family and friends, in special my mother, sisters

and father for the unconditional support in all aspects of my personal and professional life that

allowed me to complete this step in my academic development.

of 38

List of abbreviations

ALP – Alkaline phosphatase

BALP – Bone specific alkaline phosphatase

BC/CM – Breast cancer/Cancro da mama

BP – Bisphosphonates

BSP – Non collagenous bone matrix proteins: bone sialoprotein

CA15.3 – Cancer antigen 15.3

CEA – Carcinoembrionic antigen

CI – Confidence interval

CT/QT – Chemotherapy/Quimioterapia

CTX – Carboxy-terminal crosslinked telopeptide of collagen type I

CXCL12 – chemokine (C-X-C motif) ligand 12

CXCR4 – chemokine (C-X-C motif) receptor 4

CXCR7 – chemokine (C-X-C motif) receptor 7

DKK-1 – Dickkopf-1

DTC – Dessiminated tumor cells

DPD – Deoxypyridoline

ER – Estrogen receptor

HER – human epidermal growth factor receptor 2

HR – Hazard ratio

Hyl – Hydroxylysine

Hyp – Hydroxyproline

ICTP – Carboxy-terminal crosslinked telopeptide of type I collagen

MT – Marcador tumoral

NTX – Amino-terminal crosslinked telopeptide of collagen type I

OC – Osteocalcin

OP – Osteopontin

OPG – Osteoprotegerin

P1NP – Propeptides of type I procollagen

PTH-rp – Parathyroid hormone related peptide

PoM – Post-menopausal

PrM – Pre-menopausal

PYD – Pyridinoline

RANK – Receptor activator of NFƙB

RANKL – Receptor activator of NFƙB ligand

SRE – Skeletal related event

TRAP5b – Tartarate resistant acid phosphatase 5b

TRAIL – TNF-related apoptosis-inducing ligand

USA – United States of America

ZA/AZ – Zoledronic acid/Ácido zoledrónico

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